title: Structural analysis of K+ dependence in L-asparaginases from Lotus japonicus.
abstract: The molecular features responsible for the existence in plants of K+-dependent asparaginases have been investigated. For this purpose, two different cDNAs were isolated in Lotus japonicus, encoding for K+-dependent (LjNSE1) or K+-independent (LjNSE2) asparaginases. Recombinant proteins encoded by these cDNAs have been purified and characterized. Both types of asparaginases are composed by two different subunits, α (20 kDa) and β (17 kDa), disposed as (αβ)₂ quaternary structure. Major differences were found in the catalytic efficiency of both enzymes, due to the fact that K+ is able to increase by tenfold the enzyme activity and lowers the K(m) for asparagine specifically in LjNSE1 but not in LjNSE2 isoform. Optimum LjNSE1 activity was found at 5-50 mM K+, with a K(m) for K+ of 0.25 mM. Na+ and Rb+ can, to some extent, substitute for K+ on the activating effect of LjNSE1 more efficiently than Cs+ and Li+ does. In addition, K+ is able to stabilize LjNSE1 against thermal inactivation. Protein homology modelling and molecular dynamics studies, complemented with site-directed mutagenesis, revealed the key importance of E248, D285 and E286 residues for the catalytic activity and K+ dependence of LjNSE1, as well as the crucial relevance of K+ for the proper orientation of asparagine substrate within the enzyme molecule. On the other hand, LjNSE2 but not LjNSE1 showed β-aspartyl-hydrolase activity (K(m) = 0.54 mM for β-Asp-His). These results are discussed in terms of the different physiological significance of these isoenzymes in plants.
authors.affiliation: Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química, Universidad de Sevilla, 41071 Seville, Spain.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Credali
authors.forename: Antonio
authors.initials: A
authors.lastname: Díaz-Quintana
authors.forename: Margarita
authors.initials: M
authors.lastname: García-Calderón
authors.forename: Miguel A
authors.initials: MA
authors.lastname: De la Rosa
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Márquez
authors.forename: José M
authors.initials: JM
authors.lastname: Vega
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D007527:Isoenzymes; D000070116:Lotus / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D001215:Asparaginase; D009584:Nitrogen; D011188:Potassium